The Expression And Functional Identification Of A Binding Protein 53 (BP53) To White Spot Syndrome Virus

White spot syndrome virus, an attractive crustcean Whispovirus, caused a lot of losses in world aquaculture industry of shrimp since its first outbreak in 1992. To prohibit and cure the infection of WSSV is the focus now. However, methods developed for anti-WSSV infection are all in the experimental stage. The life cycle of a virus starts at its entering into the host cells by the special interaction between the receptors and the VAPs (viral attachment proteins). Now we are trying to find some attachment proteins and receptors that play a crucial role in the primary infection. Then followed we can use RNA interference, antibody prohibition, and some other bio-techniques for genomic engineering, transcriptional engineering, protein engineering, and metabolic engineering, to prevent and cure the WSSV infection effectively.VP37 was identified to be one of the VAPs of WSSV, then its binding protein BP53 was verified through affinity chromatograph and virus overlay protein binding assay (VOPBA) methods in our lab. BP53 showed a high homology with F1F0 ATP synthaseβsubunit through the analysis of MALDI-TOF MS. And partial sequence of BP53 in Litopenaeus.vannamei was achieved by method of homologous sequence clone. Based on these results, the following researches in my thesis were carried out. Three parts included in my research. The first part is the tissular location of BP53 in shrimp by both immuno-gold labeled technique and indirect immuno-fluorescent antibody technique, and the polyclonal antibody against BP53 was developed using the recombinant BP53-12.7 preparation. The second part is the temporal and spatial dynamics analysis on the expressional diversity of BP53 after challenged by WSSV. The third part is the full-length cDNA sequence cloning and the phylogenetic tree constructing of ATP synthaseβsubunit in three different species shrimps that be sensitive to WSSV.The main results in my research are described as follows: according to the bio-informatics analysis on the partial cDNA sequence of BP53, a DNA fragment of bp53 with highlight antigenicity was expressed through prokaryocyte-expression system. The recombinant expressional transformant TOP10+pBADgⅢB +bp53-12.7 were constructed subsequently. After identificated by double enzyme digestion, PCR and DNA sequence, optimization experiment was carried out by L-arc and temperature gradient induction. The ferment productions were identified by Tricine SDS-PAGE and Western-blot analysis, results indicated that the recombinant protein BP53-12.7 was expression in insoluble body and only induce under 0.02% final concentration of L-arc and 37℃can we get the maximum expression level. Recombinant protein BP53-12.7 was identified with a molecular weight of 12.7KDa. Taking DIG-WSSV as probe we had found that rBP53-12.7 had some bio-function to bind with WSSV by Western-blot analysis.The recombinant protein BP53-12.7 was separated and purified by electrophoresis. The antiserum was raised in New Zealand rabbit using purified protein rBP53-12.7. The polyclonal antibody was purified by the thalli absorption and ammonium sulphate deposition methods, then the identification of the specificity and the titer of the antiserum was carried out through Western-blot and ELISA methods. The results conformed the anti-rBP53-12.7polyclonal antibody own a good specificity against rBP53-12.7 and has a titer of 1:6400. This polyclonal antibody could be a very useful tool for localization and neutralization experiment subsequently. The localization research of BP53 is under way now.Tissue specificity expression analysis has been taken to detection of bp53 transcripts by SYBR-Green I Real-time PCR. RNA polymeraseⅡ(RPⅡ) andβactin are both taken as the internal control. On the basis of adjusting the consistent of the amplification efficiency of RPⅡ,βactin and bp53, the results of Real-time PCR can be Analysis through the detla detla CT comparative method together with Melt Curve analysis. By taken double internal control the tissue specificity expression analysis showed that the bp53 transcripts are abundant in gill and hepatocirrhosis, on the contrary, the transcripts in haemolymph and epithelial tissue are under disputed through different internal control individually. These phenomena indicate the necessity of taking many internal controls in transcripts analysis in order to decrease the errors existence in experiments. The temporary and spatial expression dynamic analysis of bp53 once shrimp infected by WSSV is doing now.Degenerate primers for homologous clone are designed by a method improved by myself, which was successful used in homologous clone of ATP synthaseβsubunit in Fenneropenaeus chinensis, Penaeus japonicus, Procambarus Clarkii. Now the whole length of Fenneropenaeus chinensis has been received while the sequence of other two shrimp both has a defection in 5'terminal. The sequence analysis of ATP synthaseβsubunit in Fenneropenaeus chinensis, Penaeus japonicus, Procambarus Clarkii, Litopenaeus.vannamei, Pacifastacus leniusculus had been taken and the results exhibit that the major diversity exist in the length of 3'UTR, while the other elements such as the TATA-box, E-box, N-terminal mitochondria target signalling sequence, AATAA tail added signalling sequence and 525aa encoded by the open reading frame are all alike. With regard to the differences existence in the cDNA and protein of ATP synthaseβsubunit, multiple sequence alignment between the five shrimps mentioned above and other WSSV non-sensitive species was carried out and corresponding phylogenenic tree was also been constructed to analysis the differences between different species. Results indicated that ATP synthaseβsubunit share high identity in shrimps, bacteria and other species individually that selected as samples for multiple sequence comparison. The major difference exists in the N-terminal and C-terminal; also there are 2 PKC Phosphorylation and 2 CK2 Phosphorylation sites existing in the C-terminal sequences.